Epigenetic control of chromatin structure has emerged as a critical layer in the complex web of interactions that regulates gene expression. Factors such as DNA methylation and histone post-translational modifications are believed to limit access of transcription factors and others effectors to their binding sites and thus, play a central role in the overall functional organization of the genome. The overall goal of this project is to further our understanding of how epigenetic information is established and maintained on globin transgenes. The significance of this goal is double. Firstly, dissecting these mechanisms will provide us with new insights into how the globin genes are regulated in their native locus. Secondly, understanding transgene silencing will help us design better gene therapy cassettes. Our experimental system will be recombinase-mediated cassette exchange (RMCE) and other Cre-based technology in mouse erythroleukemia cells and in human erythroid cells derived from human ES cells. In Aim 1, we will study the establishment of silent chromatin and try to answer the question "Do transcriptional interferences cause silencing?" We will use various methods to block transcriptional interference and the RNAi pathway. In Aim 2, we will focus on the maintenance of silent chromatin and on the question "Does DNA methylation induce a specific histone code?" We will characterize heritable and non-heritable chromatin structures using bisulfite sequencing, CHIP assays, RNAi anc targeted chromatin modifications with the help of Lac repressor fusion proteins. In Aim 3, we will extend these studies to silencing of human globin transgenes in human primary erythroid cells produced in vitro by differentiation of human ES cells